Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in the human diet and play an important role in human health. Animals are unable to synthesize carotenoids de novo and must obtain them by dietary means. Manipulation of carotenoid composition and production in plants or bacteria can provide new and/or improved sources of carotenoids. Industrial uses of carotenoids include, among others, pharmaceuticals, food supplements, animal feed additives, and colorants in cosmetics.
The genetics of carotenoid biosynthesis are well known (Armstrong, G., in Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321–352 (1999)); Lee, P. and Schmidt-Dannert, C., Appl Microbiol Biotechnol, 60:1–11 (2002); Lee et al., Chem Biol 10:453–462 (2003), and Fraser, P. and Bramley, P., Progress in Lipid Research, 43:228–265 (2004)). This pathway is extremely well studied in the Gram-negative, pigmented bacteria of the genera Pantoea, formerly known as Erwinia. Of particular interest are the genes responsible for the production of C40 carotenoids used as pigments in animal feed (e.g. canthaxanthin and astaxanthin).
The genes responsible for biosynthesis of C40 carotenoids generally can be divided into two categories: 1) the C40 carotenoid backbone biosynthesis genes responsible for the elongation, desaturation, and cyclization steps necessary for the synthesis of β-carotene (i.e. crtE, crtB, crtI, and crtY) and 2) subsequent backbone modification genes encoding enzymes involved in ketolation, hydroxylation, and glucosylation (i.e. crtW, crtO, crtZ, crtX, etc.).
A variety of methods for carotenoid production based on microbial platforms have been described in the art. Organisms such as Escherichia coli, Candida utilis, Haematococcus pluvialis, Rhodobacter sphaeroides, Paracoccus sp., and Phaffia rhodozyma have been used to produce a variety of carotenoids including, but not limited to lycopene, β-carotene, zeaxanthin, canthaxanthin, and astaxanthin (Farmer, W. R. and J. C. Liao., Biotechnol. Prog., 17: 57–61 (2001); Wang, C. et al., Biotechnol. Prog., 16: 922–926 (2000); Misawa, N. and H. Shimada., J. Biotechnol., 59:169–181 (1998); Shimada, H. et al., Appl. Environ. Microbiol., 64:2676–2680 (1998)); Albrecht, M. et al., Biotechnol. Lett., 21: 791–795 (1999); Miura, Y. et al., Appl. Environ. Microbiol., 64:1226–1229 (1998); U.S. Pat. Nos. 5,691,190; 5,466,599; 6,015,684; 5,182,208; 5,972,642; 5,656,472; 5,545,816; 5,530,189; 5,530,188; 5,429,939; 6,825,002; 5,935,808; US2004/0078846; and U.S. Pat. No. 6,124,113).
Odom et al. have demonstrated that the C1 metabolizing bacterium Methylomonas sp. 16a can be engineered for recombinant production of various C40 carotenoids (U.S. Ser. No. 09/941,947). The native C30 carotenoid biosynthetic pathway in this methylotrophic bacteria has been disrupted, creating a series of strains optimized for C40 carotenoid production (U.S. Ser. Nos. 10/997,844 and 10/997,308; hereby incorporated by reference). One of the optimized host strains, Methylomonas sp. 16a MWM1200, has been used to recombinantly produce a variety of C40 carotenoids (U.S. 60/601,947; U.S. Ser. No. 11/015,433; and U.S. 60/577,970).
Methods to increase carotenoid production in carotenogenic microbial host cells are needed to improve the economics of commercial fermentative production. One possible way to increase carotenoid production in a microbial host cell is to down-regulate and/or disrupt genes encoding enzymes involved in metabolic pathways that may compete for substrates, intermediates, and/or co-factors that influence carotenoid production.
The problem to be solved is to provide a method to increase carotenoid production in a microbial host cell by down-regulating and/or disrupting expression of one or more genes encoding enzymes involved in metabolic pathways that compete for substrates, intermediates, and/or co-factors that influence carotenoid production.